Engineering of coiled-coil protein scaffolds as innovative tools for biosensing applications
Year: 2009
Type: dissertation
Abstract: A new generation of protein scaffolds is becoming a valid alternative tool to recombinant \nantibodies of biotechnological, medical and pharmaceutical applications, where strong affinity \nand specificity are required. They share with antibodies important features (target affinity and \nspecificity), but they have also some improvements (smaller size of molecule, tolerance to \nmodification of the framework and the recognition site restricted to few residues), that can be \nexploited for biosensing application in nanotechnological platforms. Nanotechnology has been \nplayed an increasingly important role in the development of biosensors, improving the intrinsic \nfeatures of biodevices. \nIn this thesis work, we analyzed the coiled-coil domain, a widely spread dimerization \ndomain shared by several protein scaffolds, and involved in protein-protein interaction in both \neukaryotic and prokaryotic cells. The analysis of the coiled-coil structure allows a de novo \ndesign of new peptides, namely E and K, that can dimerize as a E/K coiled-coil system: the \ndimerization feature and the stability of the interaction makes this system an ideal platform to \nbuild up functional and customizable biosensors. \nA characterization of the E/K interaction was performed by using the protein \ncomplementation assay (PCA), a useful biological method to investigate the interaction between \nprotein partners. With this in vivo method, we corroborate the interaction features determinate \nwith circular dichroism, and we demonstrated that E and K coils effectively represent a protein \nscaffold, able to tolerate amino acid substitutions without altering its main structure. In addition, \nwe create two libraries of K mutant coils, randomizing the peptide sequence, and with PCA we \nselected new K binders (Kran 5.17 and Krd F8) that showed a comparable interaction activity \nwith the E-coil in preliminary in vitro tests. \nIn the last part of this work, we generate a library of a new scaffold molecule (the single \nchain E-K) capable to bind small molecules as a single protein product containing both \ndomains. Using the phage display selection system, we isolated scsE-K that can bind our \nanalyte (the caffeine) with high specificity. This new molecules can be a powerful tool for \nanalytical and biomedical applications.
Author: Tiziano Gaiotto
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W195951199